Biomarkers

ABSTRACT

The invention relates to a method of differentially diagnosing schizophrenia, bipolar disorder and major depressive disorder.

FIELD OF THE INVENTION

The invention relates to a method of differentially diagnosingschizophrenia, bipolar disorder and major depressive disorder.

BACKGROUND OF THE INVENTION Schizophrenia

Schizophrenia is a psychiatric diagnosis that describes a mentaldisorder characterized by abnormalities in the perception or expressionof reality. It most commonly manifests as auditory hallucinations,paranoid or bizarre delusions, or disorganized speech and thinking withsignificant social or occupational dysfunction. Onset of symptomstypically occurs in young adulthood, with approximately 0.4-0.6% of thepopulation affected. Diagnosis is based on the patient's self-reportedexperiences and observed behavior. No laboratory test for schizophreniacurrently exists.

Studies suggest that genetics, early environment, neurobiology,psychological and social processes are important contributory factors;some recreational and prescription drugs appear to cause or worsensymptoms. Current psychiatric research is focused on the role ofneurobiology, but no single organic cause has been found. Due to themany possible combinations of symptoms, there is debate about whetherthe diagnosis represents a single disorder or a number of discretesyndromes.

The disorder is thought to mainly affect cognition, but it also usuallycontributes to chronic problems with behavior and emotion. People withschizophrenia are likely to have additional (comorbid) conditions,including major depression and anxiety disorders; the lifetimeoccurrence of substance abuse is around 40%. Social problems, such aslong-term unemployment, poverty and homelessness, are common.Furthermore, the average life expectancy of people with the disorder is10 to 12 years less than those without, due to increased physical healthproblems and a higher suicide rate.

An important utility of biomarkers for psychotic disorders is theirresponse to medication. Administration of antipsychotics remains asubjective process, relying solely on the experience of clinicians.Furthermore, the development of antipsychotic drugs has been based onchance findings often with little relation to the background driving theobservations.

Schizophrenia is treated primarily with antipsychotic medications whichare also referred to as neuroleptic drugs or neuroleptics. Newerantipsychotic agents such as clozapine, olanzapine, quetiapine orrisperidone are thought to be more effective in improving negativesymptoms of psychotic disorders than older medication likeChlorpromazine. Furthermore, they induce less extrapyramidal sideeffects (EPS) which are movement disorders resulting from antipsychotictreatment.

The history of neuroleptics dates back to the late 19th century. Theflourishing dye industry catalyzed development of new chemicals that laythe background to modern day atypical antipsychotics. Developments inanti-malaria, anti-histamine and anaesthetic compounds also producedvarious neuroleptics. The common phenomenon to all these processes is afundamental lack of understanding of the biological mechanisms andpathways that these drugs affect, apart from the observation that theyprominently block D2 receptors in the striatum.

Bipolar Disorder

Bipolar disorder is a psychiatric disease that describes a category ofmood disorders defined by the presence of one or more episodes ofabnormally elevated mood clinically referred to as mania or, if milder,hypomania. Individuals who experience manic episodes also commonlyexperience depressive episodes or symptoms, or mixed episodes in whichfeatures of both mania and depression are present at the same time. Suchindividuals also experience a decreased quality of life. These episodesare usually separated by periods of “normal” mood, but in someindividuals, depression and mania may rapidly alternate, known as rapidcycling. Extreme manic episodes can sometimes lead to psychotic symptomssuch as delusions and hallucinations. The disorder has been subdividedinto bipolar I, bipolar II, cyclothymia, and other types, based on thenature and severity of mood episodes experienced; the range is oftendescribed as the bipolar spectrum.

Bipolar I disorder is characterised by manic episodes; the “high” of themanic-depressive cycle. Generally, this manic period is followed by aperiod of depression, although some bipolar I individuals may notexperience a major depressive episode. Mixed states, where both manic orhypomanic symptoms and depressive symptoms occur at the same time, alsooccur frequently with bipolar I patients (for example, depression withthe racing thoughts of mania). Also, dysphoric mania is common and ismania characterised by anger and irritability.

Bipolar II disorder is characterised by major depressive episodesalternating with episodes of hypomania, a milder form of mania.Hypomanic episodes can be a less disruptive form of mania and may becharacterised by low-level, non-psychotic symptoms of mania, such asincreased energy or a more elevated mood than usual. It may not affectan individual's ability to function on a day to day basis. The criteriafor hypomania differ from those for mania only by their shorter duration(at least 4 days instead of 1 week) and milder severity (no markedimpairment of functioning, hospitalisation or psychotic features).

If the depressive and manic symptoms last for two years and do not meetthe criteria for a major depressive or a manic episode then thediagnosis is classified as a cyclothymic disorder, which is a lesssevere form of bipolar affective disorder. Cyclothymic disorder isdiagnosed over the course of two years and is characterised by frequentshort periods of hypomania and depressive symptoms separated by periodsof stability.

Rapid cycling occurs when an individual's mood fluctuates fromdepression to hypomania or mania in rapid succession with little or noperiods of stability in between. One is said to experience rapid cyclingwhen one has had four or more episodes in a given year that meetcriteria for major depressive, manic, mixed or hypomanic episodes. Somepeople who rapid cycle can experience monthly, weekly or even dailyshifts in polarity (sometimes called ultra rapid cycling).

To date, no empirical diagnostic tests are available, making diagnosis asubjective evaluation which often leads to misdiagnosis and delay inaccurate treatment. When symptoms of mania, depression, mixed mood orhypomania are caused directly by a medical disorder, such as thyroiddisease or a stroke, the current diagnosis is Mood Disorder Due to aGeneral Medical Condition.

In a manic mood brought about through an antidepressant, ECT or throughan individual using street drugs, the diagnosis is Substance-InducedMood Disorder, with Manic Features.

Diagnosis of bipolar disorders has been used to categorise manicepisodes which occur as a result of taking an antidepressant medication,rather than occurring spontaneously. Confusingly, it has also been usedin instances where an individual experiences hypomania or cyclothymia(i.e. less severe mania) without major depression.

Major Depressive Disorder

Major depressive disorder is a mental disorder characterized by apervasive low mood, low self-esteem, and loss of interest or pleasure innormally enjoyable activities. The term “major depressive disorder”(which is also known as clinical depression, major depression, unipolardepression, or unipolar disorder) was selected by the AmericanPsychiatric Association for this symptom cluster under mood disorders inthe 1980 version of the Diagnostic and Statistical Manual of MentalDisorders (DSM-III) classification, and has become widely used since.

The general term depression is often used to describe the disorder, butas it is also used to describe a depressed mood, more preciseterminology is preferred in clinical and research use. Major depressionis a disabling condition which adversely affects a person's family, workor school life, sleeping and eating habits, and general health. In theUnited States, approximately 3.4% of people with major depression commitsuicide, and up to 60% of all people who commit suicide have depressionor another mood disorder.

The diagnosis of major depressive disorder is based on the patient'sself-reported experiences, behaviour reported by relatives or friends,and a mental status exam. There is no laboratory test for majordepression, although physicians generally request tests for physicalconditions that may cause similar symptoms. The most common time ofonset is between the ages of 30 and 40 years, with a later peak between50 and 60 years. Major depression is reported about twice as frequentlyin women as in men, although men are at higher risk for suicide.

Most patients are treated in the community with antidepressantmedication and some with psychotherapy or counseling. Hospitalizationmay be necessary in cases with associated self-neglect or a significantrisk of harm to self or others. A minority are treated withelectroconvulsive therapy (ECT), under a short-acting generalanaesthetic.

The course of the disorder varies widely, from one episode lastingmonths to a lifelong disorder with recurrent major depressive episodes.Depressed individuals have shorter life expectancies than those withoutdepression, in part because of greater susceptibility to medicalillnesses. Current and former patients may be stigmatized.

The understanding of the nature and causes of depression has evolvedover the centuries, though many aspects of depression remainincompletely understood and are the subject of discussion and research.

In view of the related symptoms, it is becoming increasingly difficultfor medical practitioners to effectively diagnose one psychiatricdisorder from another, particular for schizophrenia, bipolar disorderand major depressive disorder. There is therefore a pressing need forobjective molecular readouts that can differentially diagnosepsychiatric disorders such as schizophrenia, bipolar disorder and majordepressive disorder.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided the useof one or more analytes selected from: MMP 3, Alpha 1 Antitrypsin, ACTH(Adrenocorticotropic Hormone), AgRP (Agouti related Protein),Apolipoprotein A1, Apolipoprotein H, AXL, Betacellulin, BLC (BLymphocyte Chemoattractant), BDNF (Brain Derived Neurotrophic Factor),Complement 3, Cancer Antigen 125, Carcinoembryonic Antigen, CgA(Chromogranin A), Creatine Kinase MB, Cortisol, CTGF (Connective TissueGrowth Factor), EGF R, Endothelin 1, EN RAGE, Eotaxin, Epiregulin,Erythropoietin, Factor VII, Fas, Fas Ligand, Ferritin, FGF basic,Fibrinogen, FSH (Follicle Stimulating Hormone), GM CSF, GST,Haptoglobin, HB EGF, HGF (Hepatocyte growth factor), IFN gamma, IGF-1,Ig A, Ig M, IL 10, IL 12p70, IL 13, IL 15, IL 16, IL 18, IL 1alpha, IL1beta, IL 1ra, IL 2, IL 3, IL 4, IL 5, IL 7, Leptin, LH (LuteinizingHormone), Lipoprotein a, Lymphotactin, M CSF, MDC, MIP 1alpha, MIP 3alpha, MIP 1beta, Myoglobin, NrCAM, PAI 1, Prostatic Acid Phosphatase,PAPP A, PDGF, Progesterone, Prolactin, Prostate Specific Antigen Free,PARC, Peptide YY, RANTES, Resistin, S100b, Serum Amyloid P, SGOT, SHBG,SOD, sRAGE, Tamm-Horsfall Protein (THP), Thyroxine Binding Globulin,Testosterone, Tissue Factor, TECK, TIMP 1, TNF RII, TRAIL R3, ThyroidStimulating Hormone, TSP 1, VCAM 1, von Willebrand Factor, as abiomarker for the differential diagnosis of schizophrenia, bipolardisorder and major depressive disorder or predisposition thereto.

According to a second aspect of the invention, there is provided amethod of differentially diagnosing or monitoring schizophrenia, bipolardisorder and major depressive disorder, or predisposition thereto,comprising detecting and/or quantifying, in a sample from a testsubject, the analyte biomarkers defined herein. There is also provided amethod of diagnosing or monitoring major depressive disorder, orpredisposition thereto, comprising detecting and/or quantifying, in asample from a test subject, the analyte biomarkers defined herein.

According to a third aspect of the invention, there is provided a methodof differentially diagnosing schizophrenia, bipolar disorder and majordepressive disorder or predisposition thereto in an individual thereto,comprising:

-   -   (a) obtaining a biological sample from an individual;    -   (b) quantifying the amounts of one or more analyte biomarkers as        defined hereinbefore;    -   (c) comparing the amounts of the analyte biomarkers in the        biological sample with the amounts present in control biological        samples obtained from subjects having schizophrenia, bipolar        disorder and major depressive disorder to provide differential        diagnosis of schizophrenia, bipolar disorder and major        depressive disorder, or predisposition thereto. There is also        provided a method of diagnosing major depressive disorder, or        predisposition thereto, over psychotic disorders, such as        schizophrenia, in an individual, comprising:    -   (a) obtaining a biological sample from an individual;    -   (b) quantifying the amounts of the analyte biomarkers as defined        herein;    -   (c) comparing the amounts of the analyte biomarkers in the        biological sample with the amounts present in a normal control        biological sample from a normal subject, such that a difference        in the level of the analyte biomarkers in the biological sample        is indicative of major depressive disorder, or predisposition        thereto, over psychotic disorders, such as schizophrenia.

According to a fourth aspect of the invention, there is provided amethod of monitoring efficacy of a therapy in a subject having,suspected of having, or of being predisposed to major depressivedisorder, comprising detecting and/or quantifying, in a sample from saidsubject, one or more of the analyte biomarkers defined herein.

According to a fifth aspect of the invention, there is provided a methodof determining the efficacy of therapy for major depressive disorder inan individual subject comprising:

-   -   (a) obtaining a biological sample from an individual;    -   (b) quantifying the amounts of the analyte biomarkers as defined        herein;    -   (c) comparing the amounts of the analyte biomarkers in the        biological sample with the amounts present in a sample obtained        from the individual on a previous occasion, such that a        difference in the level of the analyte biomarkers in the        biological sample is indicative of a beneficial effect of the        therapy.

According to a sixth aspect of the invention, there is provided a methodof monitoring efficacy of a therapy in a subject having, suspected ofhaving, or of being predisposed to major depressive disorder, comprisingdetecting and/or quantifying, in a sample from said subject, two or moreof the second analyte biomarkers defined herein.

A further aspect of the invention provides ligands, such as naturallyoccurring or chemically synthesised compounds, capable of specificbinding to the analyte biomarker. A ligand according to the inventionmay comprise a peptide, an antibody or a fragment thereof, or an aptameror oligonucleotide, capable of specific binding to the analytebiomarker. The antibody can be a monoclonal antibody or a fragmentthereof capable of specific binding to the analyte biomarker. A ligandaccording to the invention may be labelled with a detectable marker,such as a luminescent, fluorescent or radioactive marker; alternativelyor additionally a ligand according to the invention may be labelled withan affinity tag, e.g. a biotin, avidin, streptavidin or His (e.g.hexa-His) tag.

A biosensor according to the invention may comprise the analytebiomarker or a structural/shape mimic thereof capable of specificbinding to an antibody against the analyte biomarker. Also provided isan array comprising a ligand or mimic as described herein.

Also provided by the invention is the use of one or more ligands asdescribed herein, which may be naturally occurring or chemicallysynthesised, and is suitably a peptide, antibody or fragment thereof,aptamer or oligonucleotide, or the use of a biosensor of the invention,or an array of the invention, or a kit of the invention to detect and/orquantify the analyte. In these uses, the detection and/or quantificationcan be performed on a biological sample such as from the groupconsisting of CSF, whole blood, blood serum, plasma, urine, saliva, orother bodily fluid, breath, e.g. as condensed breath, or an extract orpurification therefrom, or dilution thereof.

Diagnostic or monitoring kits are provided for performing methods of theinvention. Such kits will suitably comprise a ligand according to theinvention, for detection and/or quantification of the analyte biomarker,and/or a biosensor, and/or an array as described herein, optionallytogether with instructions for use of the kit.

A further aspect of the invention is a kit for differentially diagnosingschizophrenia, bipolar disorder and major depressive disorder, orpredisposition thereto, comprising a biosensor capable of detectingand/or quantifying one or more of the analyte biomarkers as definedherein. There is also provided a kit for monitoring or diagnosing majordepressive disorder, or predisposition thereto, over psychoticdisorders, such as schizophrenia, comprising a biosensor capable ofdetecting and/or quantifying one or more of the biomarkers as definedherein.

Biomarkers for differentially diagnosing schizophrenia, bipolar disorderand major depressive disorder are essential targets for discovery ofnovel targets and drug molecules that retard or halt progression ofthese disorders. As the level of the analyte biomarker is indicative ofdisorder and of drug response, the biomarker is useful foridentification of novel therapeutic compounds in in vitro and/or in vivoassays. Biomarkers of the invention can be employed in methods forscreening for compounds that modulate the activity of the analyte.

Thus, in a further aspect of the invention, there is provided the use ofa ligand, as described, which can be a peptide, antibody or fragmentthereof or aptamer or oligonucleotide according to the invention; or theuse of a biosensor according to the invention, or an array according tothe invention; or a kit according to the invention, to identify asubstance capable of promoting and/or of suppressing the generation ofthe biomarker.

Also there is provided a method of identifying a substance capable ofpromoting or suppressing the generation of the analyte in a subject,comprising administering a test substance to a subject animal anddetecting and/or quantifying the level of the analyte biomarker presentin a test sample from the subject.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 describes the results obtained when data from the analytes of theinvention were inputted into an algorithm to differentially diagnoseschizophrenia, bipolar disorder and major depressive disorder patients.

FIG. 2 describes a statistical analysis which indicates the optimumnumber of biomarkers for a panel of biomarkers.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided the useof one or more analytes selected from: MMP 3, Alpha 1 Antitrypsin, ACTH(Adrenocorticotropic Hormone), AgRP (Agouti related Protein),Apolipoprotein A1, Apolipoprotein H, AXL, Betacellulin, BLC (BLymphocyte Chemoattractant), BDNF (Brain Derived Neurotrophic Factor),Complement 3, Cancer Antigen 125, Carcinoembryonic Antigen, CgA(Chromogranin A), Creatine Kinase MB, Cortisol, CTGF (Connective TissueGrowth Factor), EGF R, Endothelin 1, EN RAGE, Eotaxin, Epiregulin,Erythropoietin, Factor VII, Fas, Fas Ligand, Ferritin, FGF basic,Fibrinogen, FSH (Follicle Stimulating Hormone), GM CSF, GST,Haptoglobin, HB EGF, HGF (Hepatocyte growth factor), IFN gamma, IGF-1,Ig A, Ig M, IL 10, IL 12p70, IL 13, IL 15, IL 16, IL 18, IL 1alpha, IL1beta, IL 1ra, IL 2, IL 3, IL 4, IL 5, IL 7, Leptin, LH (LuteinizingHormone), Lipoprotein a, Lymphotactin, M CSF, MDC, MIP 1alpha, MIP 3alpha, MIP 1beta, Myoglobin, NrCAM, PAI 1, Prostatic Acid Phosphatase,PAPP A, PDGF, Progesterone, Prolactin, Prostate Specific Antigen Free,PARC, Peptide YY, RANTES, Resistin, S100b, Serum Amyloid P, SGOT, SHBG,SOD, sRAGE, Tamm-Horsfall Protein (THP), Thyroxine Binding Globulin,Testosterone, Tissue Factor, TECK, TIMP 1, TNF RII, TRAIL R3, ThyroidStimulating Hormone, TSP 1, VCAM 1, von Willebrand Factor, as abiomarker for the differential diagnosis of schizophrenia, bipolardisorder and major depressive disorder or predisposition thereto.

In one embodiment, the analytes are selected from one or more of: MMP 3,Alpha 1 Antitrypsin, BLC (B Lymphocyte Chemoattractant), Cortisol,Haptoglobin, IL 16, IL 10, EGF R, Complement 3 or S100b.

In one embodiment, the analytes are selected from one or more of: MMP 3,IL 10, BDNF (Brain Derived Neurotrophic Factor), Serum Amyloid P,Betacellulin, CgA (Chromogranin A), IGF-1, S100b, IL 1ra or IL 18.

In a further embodiment, the analyte is selected from MMP-3. Data ispresented herein which demonstrates that the levels of MMP-3 were foundto be increased in patients with major depressive disorder when comparedwith schizophrenia patients and increased in patients with majordepressive disorder when compared with healthy controls. Thus, MMP-3 notonly provides a sensitive diagnostic marker for major depressivedisorder but surprisingly also provides a differential diagnostic markerfor major depressive disorder over schizophrenia.

According to a first particular aspect of the invention which may bementioned, there is provided the use of one or more analytes selectedfrom: Alpha 1 Antitrypsin, ACTH (Adrenocorticotropic Hormone), Ag RP(Agouti related Protein), Apolipoprotein A1, Apolipoprotein H, AXL,Betacellulin, BLC (B Lymphocyte Chemoattractant), BDNF (Brain DerivedNeurotrophic Factor), Complement 3, Cancer Antigen 125, CarcinoembryonicAntigen, CgA (Chromogranin A), Creatine Kinase MB, Cortisol, CTGF(Connective Tissue Growth Factor), EGF R, Endothelin 1, EN RAGE,Eotaxin, Epiregulin, Erythropoietin, Factor VII, Fas, Fas Ligand,Ferritin, FGF basic, Fibrinogen, FSH (Follicle Stimulating Hormone), GMCSF, GST, Haptoglobin, HB EGF, HGF (Hepatocyte growth factor), IFNgamma, Ig A, Ig M, IL 10, IL 12p70, IL 13, IL 15, IL 16, IL 1alpha, IL1beta, IL 1ra, IL 2, IL 3, IL 4, IL 5, IL 7, Leptin, LH (LuteinizingHormone), Lipoprotein a, Lymphotactin, M CSF, MDC, MIP 1alpha, MIP1beta, MMP 3, Myoglobin, NrCAM, PAI 1, Prostatic Acid Phosphatase, PAPPA, PDGF, Prolactin, Prostate Specific Antigen Free, PARC, Peptide YY,RANTES, Resistin, S100b, Serum Amyloid P, SGOT, SHBG, SOD, ThyroxineBinding Globulin, Testosterone, Tissue Factor, TECK, TIMP 1, TNF RII,TRAIL R3, Thyroid Stimulating Hormone, TSP 1, VCAM 1, von WillebrandFactor, as a biomarker for the differential diagnosis of schizophrenia,bipolar disorder and major depressive disorder or predispositionthereto.

It will be appreciated that the term “differential diagnosis” refers tothe positive diagnosis of a first psychiatric disorder from that of asecond psychiatric disorder. For example, in the present invention thepsychiatric disorders are selected from schizophrenia, bipolar disorderand major depressive disorder.

Thus, in one embodiment the differential diagnosis comprises diagnosisof at least one of the following conditions:

(i) schizophrenia from bipolar disorder;(ii) schizophrenia from major depressive disorder;(iii) schizophrenia from bipolar disorder and major depressive disorder;(iv) bipolar disorder from schizophrenia;(v) bipolar disorder from major depressive disorder;(vi) bipolar disorder from schizophrenia and major depressive disorder;(vii) major depressive disorder from schizophrenia;(viii) major depressive disorder from bipolar disorder; or(ix) major depressive disorder from schizophrenia and bipolar disorder.

In a further embodiment, the differential diagnosis comprises diagnosisof at least one of the following conditions:

(iii) schizophrenia from normal controls and bipolar disorder and majordepressive disorder;(vi) bipolar disorder from normal controls and schizophrenia and majordepressive disorder; or(ix) major depressive disorder from normal controls and schizophreniaand bipolar disorder.

According to a second particular aspect of the invention which may bementioned, there is provided the use of one or more analytes selectedfrom: Tamm-Horsfall Protein (THP), MIP-3 alpha Betacellulin, MMP-3,sRAGE, IL-1ra, Progesterone, IL-10, Brain Derived Neurotrophic Factor(BDNF), Serum Amyloid P, Chromogranin A, Creatine Kinase MB, IGF-1,S100b and IL-18, as a biomarker for the differential diagnosis of majordepressive disorder, or predisposition thereto, over psychoticdisorders, such as schizophrenia.

Data is presented herein which shows that the levels of the 15 analytesof the second particular aspect of the invention were found to besignificantly altered between the major depressive disorder group andthe schizophrenia group (Table 3). The invention therefore provides aspecific and sensitive diagnosis for major depressive disorder which maybe differentiated from psychotic disorders such as schizophrenia.

In one embodiment of the second particular aspect of the invention, theanalyte is selected from MMP-3, Betacellulin, MIP-3 alpha andTamm-Horsfall Protein (THP). Data is presented herein which demonstratesthat the biomarkers of this embodiment were found to be increased inpatients with major depressive disorder when compared with schizophreniapatients (for example the data shows a fold change of >1).

In a further embodiment of the second particular aspect of theinvention, the analyte is selected from MMP-3. Data is presented hereinwhich demonstrates that the levels of MMP-3 were found to be increasedin patients with major depressive disorder when compared withschizophrenia patients and increased in patients with major depressivedisorder when compared with healthy controls. Thus, MMP-3 not onlyprovides a sensitive diagnostic marker for major depressive disorder butsurprisingly also provides a differential diagnostic marker for majordepressive disorder over schizophrenia. Thus, according to a furtheraspect of the invention there is provided the use of MMP-3 as abiomarker for major depressive disorder, or predisposition thereto. Inone embodiment of this aspect of the invention, the use additionallycomprises one or more analytes selected from sRAGE, Betacellulin, MIP-3alpha and Tamm-Horsfall Protein (THP).

In one embodiment of the second particular aspect of the invention, theanalyte is selected from sRAGE. Data is presented herein whichdemonstrates that the biomarker of this embodiment was found to bedecreased in patients with major depressive disorder when compared withschizophrenia patients (for example the data shows a fold change of <1).

In one embodiment of the second particular aspect of the invention, theanalyte is selected from: IL-1ra, Progesterone, IL-10, Serum Amyloid P,Chromogranin A, Creatine Kinase MB, S100b and IL-18. Data is presentedherein which demonstrates that the biomarkers of this embodiment werefound to be increased in patients with major depressive disorder whencompared with schizophrenia patients.

In a further embodiment of the second particular aspect of theinvention, the analyte is selected from IL-1ra. Data is presented hereinwhich demonstrates that the levels of IL-1ra were found to be increasedin patients with major depressive disorder when compared withschizophrenia patients and increased in patients with major depressivedisorder when compared with healthy controls. Thus, IL-1ra not onlyprovides a sensitive diagnostic marker for major depressive disorder butsurprisingly also provides a differential diagnostic marker for majordepressive disorder over schizophrenia. Thus, according to a furtheraspect of the invention, there is provided the use of IL-1ra incombination with one or more analytes selected from MMP-3, sRAGE,Betacellulin, MIP-3 alpha, Tamm-Horsfall Protein (THP), Progesterone,IL-10, Brain Derived Neurotrophic Factor (BDNF), Serum Amyloid P,Chromogranin A, Creatine Kinase MB, IGF-1, S100b and IL-18, as abiomarker for major depressive disorder, or predisposition thereto. Inone embodiment of this aspect of the invention, the analyte is selectedfrom MMP-3. According to a further aspect of the invention, there isprovided the use of MMP-3 and IL-1ra as a specific panel of analytebiomarkers for the differential diagnosis of major depressive disorder,or predisposition thereto over psychotic disorders, such asschizophrenia. In one embodiment of this aspect of the invention, thepanel additionally comprises one or more analytes selected from sRAGE,Betacellulin, MIP-3 alpha, Tamm-Horsfall Protein (THP), Progesterone,IL-10, Brain Derived Neurotrophic Factor (BDNF), Serum Amyloid P,Chromogranin A, Creatine Kinase MB, IGF-1, S100b and IL-18.

In an alternative embodiment of the second particular aspect of theinvention, the analyte is selected from: Brain Derived NeurotrophicFactor (BDNF) and IGF-1. Data is presented herein which demonstratesthat the biomarkers of this embodiment were found to be decreased inpatients with major depressive disorder when compared with schizophreniapatients.

According to a further aspect of the invention, there is provided theuse of MMP-3, sRAGE, Betacellulin, MIP-3 alpha, Tamm-Horsfall Protein(THP), IL-1ra, Progesterone, IL-10, Brain Derived Neurotrophic Factor(BDNF), Serum Amyloid P, Chromogranin A, Creatine Kinase MB, IGF-1,S100b and IL-18 as a specific panel of analyte biomarkers for majordepressive disorder, or predisposition thereto. Data is presented hereinwhich demonstrates that this specific panel of biomarkers were found tobe altered in patients with major depressive disorder when compared withschizophrenia patients. Thus, according to a further aspect of theinvention, there is provided the use of MMP-3, sRAGE, Betacellulin,MIP-3 alpha, Tamm-Horsfall Protein (THP), IL-1ra, Progesterone, IL-10,Brain Derived Neurotrophic Factor (BDNF), Serum Amyloid P, ChromograninA, Creatine Kinase MB, IGF-1, S100b and IL-18 as a specific panel ofanalyte biomarkers for the differential diagnosis of major depressivedisorder, or predisposition thereto over psychotic disorders, such asschizophrenia.

According to a further aspect of the invention, there is provided theuse of MMP-3, Betacellulin, MIP-3 alpha, Tamm-Horsfall Protein (THP),IL-1ra, Progesterone, IL-10, Serum Amyloid P, Chromogranin A, CreatineKinase MB, S100b and IL-18 as a specific panel of analyte biomarkers formajor depressive disorder, or predisposition thereto. Data is presentedherein which demonstrates that this specific panel of biomarkers werefound to be increased in patients with major depressive disorder whencompared with schizophrenia patients. Thus, according to a furtheraspect of the invention, there is provided the use of MMP-3,Betacellulin, MIP-3 alpha, Tamm-Horsfall Protein (THP), IL-1ra,Progesterone, IL-10, Serum Amyloid P, Chromogranin A, Creatine KinaseMB, S100b and IL-18 as a specific panel of analyte biomarkers for thedifferential diagnosis of major depressive disorder, or predispositionthereto over psychotic disorders, such as schizophrenia.

According to a further aspect of the invention, there is provided theuse of Brain Derived Neurotrophic Factor (BDNF), sRAGE and IGF-1 as aspecific panel of analyte biomarkers for major depressive disorder, orpredisposition thereto. Data is presented herein which demonstrates thatthis specific panel of biomarkers were found to be decreased in patientswith major depressive disorder when compared with schizophreniapatients. Thus, according to a further aspect of the invention, there isprovided the use of Brain Derived Neurotrophic Factor (BDNF), sRAGE andIGF-1 as a specific panel of analyte biomarkers for the differentialdiagnosis of major depressive disorder, or predisposition thereto overpsychotic disorders, such as schizophrenia. The term “biomarker” means adistinctive biological or biologically derived indicator of a process,event, or condition. Peptide biomarkers can be used in methods ofdiagnosis, e.g. clinical screening, and prognosis assessment and inmonitoring the results of therapy, identifying patients most likely torespond to a particular therapeutic treatment, drug screening anddevelopment. Biomarkers and uses thereof are valuable for identificationof new drug treatments and for discovery of new targets for drugtreatment.

In one embodiment of the invention, the number of analytes comprise anyone of the following numbers of analytes: 2 or more, 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more,11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more,17 or more, 18 or more, 19 or more or 20 or more. Results of astatistical analysis with a known algorithm are shown in FIG. 2 whereinit can be seen that the most predictive analyte achieved aclassification accuracy rate of 0.74022. The results of additionalanalyte biomarkers were then added to the algorithm to improvestatistical performance until it can no longer be improved. It can beseen from the results shown in FIG. 2 that a panel of at least 5biomarkers achieved a correctness rate of greater than 0.77 and that theperformance did not improve for a panel of biomarkers in excess of 15.The results of the analysis shown in FIG. 2 therefore demonstrate thatthe optimal panel size for the analyte biomarkers of the invention isbetween 5 and 15. Thus, in one embodiment, there is provided a panel ofbiomarkers which comprises between 5 and 15 of the analytes hereinbeforedefined.

According to a further aspect of the invention, there is provided amethod of differentially diagnosing schizophrenia, bipolar disorder andmajor depressive disorder or predisposition thereto in an individualthereto, comprising:

-   -   (a) obtaining a biological sample from an individual;    -   (b) quantifying the amounts of one or more analyte biomarkers as        defined hereinbefore;    -   (c) comparing the amounts of the analyte biomarkers in the        biological sample with the amounts present in control biological        samples obtained from subjects having schizophrenia, bipolar        disorder and major depressive disorder to provide differential        diagnosis of schizophrenia, bipolar disorder and major        depressive disorder, or predisposition thereto.

According to a further aspect of the invention, there is provided amethod of diagnosing major depressive disorder, or predispositionthereto, in an individual thereto comprising

-   -   a) obtaining a biological sample from an individual;    -   b) quantifying the amounts of a panel of analyte biomarkers in        the biological sample, wherein the panel of analyte biomarkers        comprises Brain Derived Neurotrophic Factor (BDNF), sRAGE and        IGF-1; and    -   c) comparing the amounts of the panel of analyte biomarkers in        the biological sample with the amounts present in a normal        control biological sample from a normal subject, wherein a lower        level of the panel of analyte biomarkers in the biological        sample is indicative of major depressive disorder, or        predisposition thereto.

In one embodiment, the lower level is a <1 fold difference relative tothe control sample, such as a fold difference of 0.9, 0.8, 0.7, 0.6,0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 or any ranges therebetween. In oneembodiment, the lower level is between 0.1 and 0.9 fold differencerelative to the control sample, such as between 0.5 and 0.9.

According to a further aspect of the invention, there is provided amethod of diagnosing major depressive disorder, or predispositionthereto, in an individual thereto comprising

-   -   a) obtaining a biological sample from an individual;    -   b) quantifying the amounts of a panel of analyte biomarkers in        the biological sample, wherein the panel of analyte biomarkers        comprises MMP-3, Betacellulin, MIP-3 alpha, Tamm-Horsfall        Protein (THP), IL-1ra, Progesterone, IL-10, Serum Amyloid P,        Chromogranin A, Creatine Kinase MB, S100b and IL-18; and    -   c) comparing the amounts of the panel of analyte biomarkers in        the biological sample with the amounts present in a normal        control biological sample from a normal subject, wherein a        higher level of the panel of analyte biomarkers in the        biological sample is indicative of major depressive disorder, or        predisposition thereto.

In one embodiment, the higher level is a >1 fold difference relative tothe control sample, such as a fold difference of 1.1, 1.2, 1.3, 1.4,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15 or 20 or any rangestherebetween. In one embodiment, the higher level is between 1 and 10fold difference relative to the control sample, such as between 1.2 and2.5.

As used herein, the term “biosensor” means anything capable of detectingthe presence of the biomarker. Examples of biosensors are describedherein.

In one embodiment, one or more of the biomarkers defined hereinbeforemay be replaced by a molecule, or a measurable fragment of the molecule,found upstream or downstream of the biomarker in a biological pathway.

Biosensors according to the invention may comprise a ligand or ligands,as described herein, capable of specific binding to the analytebiomarker. Such biosensors are useful in detecting and/or quantifying ananalyte of the invention.

Diagnostic kits for the differential diagnosis of schizophrenia, bipolardisorder and major depressive disorder or predisposition thereto aredescribed herein. In one embodiment, the kits additionally contain abiosensor capable of detecting and/or quantifying a peptide biomarker.

In methods of diagnosing according to the invention, detecting and/orquantifying the peptide biomarker in a biological sample from a testsubject may be performed on two or more occasions. Comparisons may bemade between the level of biomarker in samples taken on two or moreoccasions. Assessment of any change in the level of the peptidebiomarker in samples taken on two or more occasions may be performed.Modulation of the analyte biomarker level is useful as an indicator ofthe state of schizophrenia, bipolar disorder and major depressivedisorder or predisposition thereto. An increase in the level of thebiomarker, over time is indicative of onset or progression, i.e.worsening of this disorder, whereas a decrease in the level of theanalyte biomarker indicates amelioration or remission of the disorder,or vice versa.

A method of diagnosis according to the invention may comprisequantifying the analyte biomarker in a test biological sample from atest subject and comparing the level of the analyte present in said testsample with one or more controls.

The control used in a method of the invention can be one or morecontrol(s) selected from the group consisting of: the level in a samplefrom a subject with schizophrenia, bipolar disorder and major depressivedisorder, or a diagnosed predisposition thereto; schizophrenia, bipolardisorder or major depressive disorder biomarker analyte level, orschizophrenia, bipolar disorder and major depressive disorder biomarkeranalyte range.

In one embodiment, there is provided a method of diagnosing majordepressive disorder, or predisposition thereto, which comprises:

-   -   (a) quantifying the amount of the peptide biomarker in a test        biological sample; and    -   (b) comparing the amount of said peptide in said test sample        with the amount present in a normal control biological sample        from a normal subject.

For biomarkers which are increased in patients with major depressivedisorder, a higher level of the peptide biomarker in the test samplerelative to the level in the normal control is indicative of thepresence of major depressive disorder, or predisposition thereto; anequivalent or lower level of the peptide in the test sample relative tothe normal control is indicative of absence of major depressive disorderand/or absence of a predisposition thereto. For biomarkers which aredecreased in patients with major depressive disorder, a lower level ofthe peptide biomarker in the test sample relative to the level in thenormal control is indicative of the presence of major depressivedisorder, or predisposition thereto; an equivalent or lower level of thepeptide in the test sample relative to the normal control is indicativeof absence of major depressive disorder and/or absence of apredisposition thereto.

The term “diagnosis” as used herein encompasses identification,confirmation, and/or characterisation of schizophrenia, bipolar disorderand major depressive disorder, or predisposition thereto. Bypredisposition it is meant that a subject does not currently presentwith the disorder, but is liable to be affected by the disorder in time.Methods of diagnosis according to the invention are useful to confirmthe existence of a disorder, or predisposition thereto. Methods ofdiagnosis are also useful in methods for assessment of clinicalscreening, prognosis, choice of therapy, evaluation of therapeuticbenefit, i.e. for drug screening and drug development.

Efficient diagnosis methods provide very powerful “patient solutions”with the potential for improved prognosis, by establishing the correctdiagnosis, allowing rapid identification of the most appropriatetreatment (thus lessening unnecessary exposure to harmful drug sideeffects), reducing “down-time” and relapse rates.

Also provided is a method of monitoring efficacy of a therapy for majordepressive disorder in a subject having such a disorder, suspected ofhaving such a disorder, or of being predisposed thereto, comprisingdetecting and/or quantifying the peptide present in a biological samplefrom said subject. In monitoring methods, test samples may be taken ontwo or more occasions. The method may further comprise comparing thelevel of the biomarker(s) present in the test sample with one or morecontrol(s) and/or with one or more previous test sample(s) taken earlierfrom the same test subject, e.g. prior to commencement of therapy,and/or from the same test subject at an earlier stage of therapy. Themethod may comprise detecting a change in the level of the biomarker(s)in test samples taken on different occasions.

The invention provides a method for monitoring efficacy of therapy formajor depressive disorder in a subject, comprising:

-   -   (a) quantifying the amount of the peptide biomarker; and    -   (b) comparing the amount of said peptide in said test sample        with the amount present in one or more control(s) and/or one or        more previous test sample(s) taken at an earlier time from the        same test subject.

For biomarkers which are increased in patients with major depressivedisorder, a decrease in the level of the peptide biomarker in the testsample relative to the level in a previous test sample taken earlierfrom the same test subject is indicative of a beneficial effect, e.g.stabilisation or improvement, of said therapy on the disorder, suspecteddisorder or predisposition thereto. For biomarkers which are decreasedin patients with major depressive disorder, an increase in the level ofthe peptide biomarker in the test sample relative to the level in aprevious test sample taken earlier from the same test subject isindicative of a beneficial effect, e.g. stabilisation or improvement, ofsaid therapy on the disorder, suspected disorder or predispositionthereto.

Methods for monitoring efficacy of a therapy can be used to monitor thetherapeutic effectiveness of existing therapies and new therapies inhuman subjects and in non-human animals (e.g. in animal models). Thesemonitoring methods can be incorporated into screens for new drugsubstances and combinations of substances.

Suitably, the time elapsed between taking samples from a subjectundergoing diagnosis or monitoring will be 3 days, 5 days, a week, twoweeks, a month, 2 months, 3 months, 6 or 12 months. Samples may be takenprior to and/or during and/or following an anti-depressant therapy.Samples can be taken at intervals over the remaining life, or a partthereof, of a subject.

The term “detecting” as used herein means confirming the presence of theanalyte biomarker present in the sample. Quantifying the amount of theanalyte biomarker present in a sample may include determining theconcentration of the analyte biomarker present in the sample. Detectingand/or quantifying may be performed directly on the sample, orindirectly on an extract therefrom, or on a dilution thereof.

In alternative aspects of the invention, the presence of the analytebiomarker is assessed by detecting and/or quantifying antibody orfragments thereof capable of specific binding to the biomarker that aregenerated by the subject's body in response to the analyte and thus arepresent in a biological sample from a subject having schizophrenia,bipolar disorder or major depressive disorder or a predispositionthereto.

Detecting and/or quantifying can be performed by any method suitable toidentify the presence and/or amount of a specific analyte in abiological sample from a patient or a purification or extract of abiological sample or a dilution thereof. In methods of the invention,quantifying may be performed by measuring the concentration of theanalyte biomarker in the sample or samples. Biological samples that maybe tested in a method of the invention include cerebrospinal fluid(CSF), whole blood, blood serum, plasma, urine, saliva, or other bodilyfluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. ascondensed breath, or an extract or purification therefrom, or dilutionthereof. Biological samples also include tissue homogenates, tissuesections and biopsy specimens from a live subject, or taken post-mortem.The samples can be prepared, for example where appropriate diluted orconcentrated, and stored in the usual manner.

Detection and/or quantification of analyte biomarkers may be performedby detection of the analyte biomarker or of a fragment thereof, e.g. afragment with C-terminal truncation, or with N-terminal truncation.Fragments are suitably greater than 4 amino acids in length, for example5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsin length.

The biomarker may be directly detected, e.g. by SELDI or MALDI-TOF.Alternatively, the biomarker may be detected directly or indirectly viainteraction with a ligand or ligands such as an antibody or abiomarker-binding fragment thereof, or other peptide, or ligand, e.g.aptamer, or oligonucleotide, capable of specifically binding thebiomarker. The ligand may possess a detectable label, such as aluminescent, fluorescent or radioactive label, and/or an affinity tag.

For example, detecting and/or quantifying can be performed by one ormore method(s) selected from the group consisting of: SELDI (-TOF),MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Massspec (MS), reverse phase (RP) LC, size permeation (gel filtration), ionexchange, affinity, HPLC, UPLC and other LC or LC MS-based techniques.Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA,USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography(e.g. high pressure liquid chromatography (HPLC) or low pressure liquidchromatography (LPLC)), thin-layer chromatography, NMR (nuclear magneticresonance) spectroscopy could also be used.

Methods of diagnosing according to the invention may comprise analysinga sample of cerebrospinal fluid (CSF) by SELDI TOF or MALDI TOF todetect the presence or level of the analyte biomarker. These methods arealso suitable for clinical screening, prognosis, monitoring the resultsof therapy, identifying patients most likely to respond to a particulartherapeutic treatment, for drug screening and development, andidentification of new targets for drug treatment.

Detecting and/or quantifying the analyte biomarkers may be performedusing an immunological method, involving an antibody, or a fragmentthereof capable of specific binding to the analyte biomarker. Suitableimmunological methods include sandwich immunoassays, such as sandwichELISA, in which the detection of the analyte biomarkers is performedusing two antibodies which recognize different epitopes on a analytebiomarker; radioimmunoassays (RIA), direct, indirect or competitiveenzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA),Fluorescence immunoassays (FIA), western blotting, immunoprecipitationand any particle-based immunoassay (e.g. using gold, silver, or latexparticles, magnetic particles, or Q-dots). Immunological methods may beperformed, for example, in microtitre plate or strip format.

Immunological methods in accordance with the invention may be based, forexample, on any of the following methods.

Immunoprecipitation is the simplest immunoassay method; this measuresthe quantity of precipitate, which forms after the reagent antibody hasincubated with the sample and reacted with the target antigen presenttherein to form an insoluble aggregate. Immunoprecipitation reactionsmay be qualitative or quantitative.

In particle immunoassays, several antibodies are linked to the particle,and the particle is able to bind many antigen molecules simultaneously.This greatly accelerates the speed of the visible reaction. This allowsrapid and sensitive detection of the biomarker.

In immunonephelometry, the interaction of an antibody and target antigenon the biomarker results in the formation of immune complexes that aretoo small to precipitate. However, these complexes will scatter incidentlight and this can be measured using a nephelometer. The antigen, i.e.biomarker, concentration can be determined within minutes of thereaction.

Radioimmunoassay (RIA) methods employ radioactive isotopes such as I¹²⁵to label either the antigen or antibody. The isotope used emits gammarays, which are usually measured following removal of unbound (free)radiolabel. The major advantages of RIA, compared with otherimmunoassays, are higher sensitivity, easy signal detection, andwell-established, rapid assays. The major disadvantages are the healthand safety risks posed by the use of radiation and the time and expenseassociated with maintaining a licensed radiation safety and disposalprogram. For this reason, RIA has been largely replaced in routineclinical laboratory practice by enzyme immunoassays.

Enzyme (EIA) immunoassays were developed as an alternative toradioimmunoassays (RIA). These methods use an enzyme to label either theantibody or target antigen. The sensitivity of EIA approaches that forRIA, without the danger posed by radioactive isotopes. One of the mostwidely used EIA methods for detection is the enzyme-linked immunosorbentassay (ELISA). ELISA methods may use two antibodies one of which isspecific for the target antigen and the other of which is coupled to anenzyme, addition of the substrate for the enzyme results in productionof a chemiluminescent or fluorescent signal.

Fluorescent immunoassay (FIA) refers to immunoassays which utilize afluorescent label or an enzyme label which acts on the substrate to forma fluorescent product. Fluorescent measurements are inherently moresensitive than colorimetric (spectrophotometric) measurements.Therefore, FIA methods have greater analytical sensitivity than EIAmethods, which employ absorbance (optical density) measurement.

Chemiluminescent immunoassays utilize a chemiluminescent label, whichproduces light when excited by chemical energy; the emissions aremeasured using a light detector.

Immunological methods according to the invention can thus be performedusing well-known methods. Any direct (e.g., using a sensor chip) orindirect procedure may be used in the detection of peptide biomarkers ofthe invention.

The Biotin-Avidin or Biotin-Streptavidin systems are generic labellingsystems that can be adapted for use in immunological methods of theinvention. One binding partner (hapten, antigen, ligand, aptamer,antibody, enzyme etc) is labelled with biotin and the other partner(surface, e.g. well, bead, sensor etc) is labelled with avidin orstreptavidin. This is conventional technology for immunoassays, geneprobe assays and (bio)sensors, but is an indirect immobilisation routerather than a direct one. For example a biotinylated ligand (e.g.antibody or aptamer) specific for an analyte biomarker of the inventionmay be immobilised on an avidin or streptavidin surface, the immobilisedligand may then be exposed to a sample containing or suspected ofcontaining the analyte biomarker in order to detect and/or quantify ananalyte biomarker of the invention. Detection and/or quantification ofthe immobilised antigen may then be performed by an immunological methodas described herein.

The term “antibody” as used herein includes, but is not limited to:polyclonal, monoclonal, bispecific, humanised or chimeric antibodies,single chain antibodies, Fab fragments and F(ab′)₂ fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies and epitope-binding fragments of any of the above. The term“antibody” as used herein also refers to immunoglobulin molecules andimmunologically-active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bindsan antigen. The immunoglobulin molecules of the invention can be of anyclass (e.g., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulinmolecule.

The identification of key biomarkers specific to a disease is central tointegration of diagnostic procedures and therapeutic regimes. Usingpredictive biomarkers appropriate diagnostic tools such as biosensorscan be developed, accordingly, in methods and uses of the invention,detecting and quantifying can be performed using a biosensor,microanalytical system, microengineered system, microseparation system,immunochromatography system or other suitable analytical devices. Thebiosensor may incorporate an immunological method for detection of thebiomarker(s), electrical, thermal, magnetic, optical (e.g. hologram) oracoustic technologies. Using such biosensors, it is possible to detectthe target biomarker(s) at the anticipated concentrations found inbiological samples.

Thus, according to a further aspect of the invention there is providedan apparatus for differentially diagnosing schizophrenia, bipolardisorder and major depressive disorder or predisposition thereto whichcomprises a biosensor, microanalytical, microengineered, microseparationand/or immunochromatography system configured to detect and/or quantifyany of the biomarkers defined herein.

The biomarker(s) of the invention can be detected using a biosensorincorporating technologies based on “smart” holograms, or high frequencyacoustic systems, such systems are particularly amenable to “bar code”or array configurations.

In smart hologram sensors (Smart Holograms Ltd, Cambridge, UK), aholographic image is stored in a thin polymer film that is sensitised toreact specifically with the biomarker. On exposure, the biomarker reactswith the polymer leading to an alteration in the image displayed by thehologram. The test result read-out can be a change in the opticalbrightness, image, colour and/or position of the image. For qualitativeand semi-quantitative applications, a sensor hologram can be read byeye, thus removing the need for detection equipment. A simple coloursensor can be used to read the signal when quantitative measurements arerequired. Opacity or colour of the sample does not interfere withoperation of the sensor. The format of the sensor allows multiplexingfor simultaneous detection of several substances. Reversible andirreversible sensors can be designed to meet different requirements, andcontinuous monitoring of a particular biomarker of interest is feasible.

Suitably, biosensors for detection of one or more biomarkers of theinvention combine biomolecular recognition with appropriate means toconvert detection of the presence, or quantitation, of the biomarker inthe sample into a signal.

Biosensors can be adapted for “alternate site” diagnostic testing, e.g.in the ward, outpatients' department, surgery, home, field andworkplace.

Biosensors to detect one or more biomarkers of the invention includeacoustic, plasmon resonance, holographic and microengineered sensors.Imprinted recognition elements, thin film transistor technology,magnetic acoustic resonator devices and other novel acousto-electricalsystems may be employed in biosensors for detection of the one or morebiomarkers of the invention.

Methods involving detection and/or quantification of one or more analytebiomarkers of the invention can be performed on bench-top instruments,or can be incorporated onto disposable, diagnostic or monitoringplatforms that can be used in a non-laboratory environment, e.g. in thephysician's office or at the patient's bedside. Suitable biosensors forperforming methods of the invention include “credit” cards with opticalor acoustic readers. Biosensors can be configured to allow the datacollected to be electronically transmitted to the physician forinterpretation and thus can form the basis for e-neuromedicine.

Any suitable animal may be used as a subject non-human animal, forexample a non-human primate, horse, cow, pig, goat, sheep, dog, cat,fish, rodent, e.g. guinea pig, rat or mouse; insect (e.g. Drosophila),amphibian (e.g. Xenopus) or C. elegans.

The test substance can be a known chemical or pharmaceutical substance,such as, but not limited to, an anti-depressive disorder therapeutic; orthe test substance can be novel synthetic or natural chemical entity, ora combination of two or more of the aforesaid substances.

There is provided a method of identifying a substance capable ofpromoting or suppressing the generation of the analyte biomarker in asubject, comprising exposing a test cell to a test substance andmonitoring the level of the analyte biomarker within said test cell, orsecreted by said test cell.

The test cell could be prokaryotic, however a eukaryotic cell willsuitably be employed in cell-based testing methods. Suitably, theeukaryotic cell is a yeast cell, insect cell, Drosophila cell, amphibiancell (e.g. from Xenopus), C. elegans cell or is a cell of human,non-human primate, equine, bovine, porcine, caprine, ovine, canine,feline, piscine, rodent or murine origin.

In methods for identifying substances of potential therapeutic use,non-human animals or cells can be used that are capable of expressingthe peptide.

Screening methods also encompass a method of identifying a ligandcapable of binding to the analyte biomarker according to the invention,comprising incubating a test substance in the presence of the analytebiomarker in conditions appropriate for binding, and detecting and/orquantifying binding of the analyte to said test substance.

High-throughput screening technologies based on the biomarker, uses andmethods of the invention, e.g. configured in an array format, aresuitable to monitor biomarker signatures for the identification ofpotentially useful therapeutic compounds, e.g. ligands such as naturalcompounds, synthetic chemical compounds (e.g. from combinatoriallibraries), peptides, monoclonal or polyclonal antibodies or fragmentsthereof, which may be capable of binding the biomarker.

Methods of the invention can be performed in array format, e.g. on achip, or as a multiwell array. Methods can be adapted into platforms forsingle tests, or multiple identical or multiple non-identical tests, andcan be performed in high throughput format. Methods of the invention maycomprise performing one or more additional, different tests to confirmor exclude diagnosis, and/or to further characterise a condition.

The invention further provides a substance, e.g. a ligand, identified oridentifiable by an identification or screening method or use of theinvention. Such substances may be capable of inhibiting, directly orindirectly, the activity of the analyte biomarker, or of suppressinggeneration of the analyte biomarker.

The term “substances” includes substances that do not directly bind thepeptide biomarker and directly modulate a function, but insteadindirectly modulate a function of the analyte biomarker. Ligands arealso included in the term substances; ligands of the invention (e.g. anatural or synthetic chemical compound, peptide, aptamer,oligonucleotide, antibody or antibody fragment) are capable of binding,suitably specific binding, to the analyte.

The invention further provides a substance according to the inventionfor use in the treatment of schizophrenia, bipolar disorder or majordepressive disorder or predisposition thereto. For example, theinvention provides a substance according to the invention for use in thetreatment of major depressive disorder, or predisposition thereto.

Also provided is the use of a substance according to the invention inthe treatment of schizophrenia, bipolar disorder or major depressivedisorder, or predisposition thereto. For example, also provided is theuse of a substance according to the invention in the treatment of majordepressive disorder, or predisposition thereto.

Also provided is the use of a substance according to the invention as amedicament.

Yet further provided is the use of a substance according to theinvention in the manufacture of a medicament for the treatment of majordepressive disorder, or predisposition thereto.

A kit for differentially diagnosing schizophrenia, bipolar disorder ormajor depressive disorder, or predisposition thereto is provided. A kitfor diagnosing or monitoring major depressive disorder, orpredisposition thereto is also provided. Suitably a kit according to theinvention may contain one or more components selected from the group: aligand specific for the analyte biomarker or a structural/shape mimic ofthe analyte biomarker, one or more controls, one or more reagents andone or more consumables; optionally together with instructions for useof the kit in accordance with any of the methods defined herein.

The identification of biomarkers for differentially diagnosingschizophrenia, bipolar disorder or major depressive disorder permitsintegration of diagnostic procedures and therapeutic regimes. Currentlythere are significant delays in determining effective treatment andhitherto it has not been possible to perform rapid assessment of drugresponse. Traditionally, many psychiatric therapies have requiredtreatment trials lasting weeks to months for a given therapeuticapproach. Detection of an analyte biomarker of the invention can be usedto screen subjects prior to their participation in clinical trials. Thebiomarkers provide the means to indicate therapeutic response, failureto respond, unfavourable side-effect profile, degree of medicationcompliance and achievement of adequate serum drug levels. The biomarkersmay be used to provide warning of adverse drug response. Biomarkers areuseful in development of personalized brain therapies, as assessment ofresponse can be used to fine-tune dosage, minimise the number ofprescribed medications, reduce the delay in attaining effective therapyand avoid adverse drug reactions. Thus by monitoring a biomarker of theinvention, patient care can be tailored precisely to match the needsdetermined by the disorder and the pharmacogenomic profile of thepatient, the biomarker can thus be used to titrate the optimal dose,predict a positive therapeutic response and identify those patients athigh risk of severe side effects.

Biomarker-based tests provide a first line assessment of ‘new’ patients,and provide objective measures for accurate and rapid diagnosis, in atime frame and with precision, not achievable using the currentsubjective measures.

Furthermore, diagnostic biomarker tests are useful to identify familymembers or patients at high risk of developing schizophrenia, bipolardisorder or major depressive disorder. This permits initiation ofappropriate therapy, or preventive measures, e.g. managing risk factors.These approaches are recognised to improve outcome and may prevent overtonset of the disorder.

Biomarker monitoring methods, biosensors and kits are also vital aspatient monitoring tools, to enable the physician to determine whetherrelapse is due to worsening of the disorder, poor patient compliance orsubstance abuse. If pharmacological treatment is assessed to beinadequate, then therapy can be reinstated or increased; a change intherapy can be given if appropriate. As the biomarkers are sensitive tothe state of the disorder, they provide an indication of the impact ofdrug therapy or of substance abuse.

The following studies illustrate the invention.

Example 1 Methodology

(A) Raw Data Acquisition 144 analytes were measured in the serum of 653patients with schizophrenia (SZ), bipolar disorder (BD) or majordepressive disorder (MDD) using multiplexed immunoassays which may beperformed in accordance with known procedures. All samples were analyzedin a randomized and blinded manner.

(B) Data Pre-Analysis

Available metadata was investigated to estimate its relationship to theoutcome variable of interest (e.g. pair-wise difference between alldisease groups) in order to identify potential sources of selectionbias. Also, outcome as well as molecular variables were inspectedvisually and statistically to identify underlying distributions andpotential justifications for future data transformation ornormalization. The relationship between meta-data and molecular data wasexamined to identify potential confounding factors and covariates.

(C) Data Processing

Data processing consisted of three steps:

(i) Missing Value Imputation

Values measured below the lower limit of detection were replaced by halfthe minimum value measured for a given analyte. Values higher than theupper limit of detection were replaced by the upper limit of detectionfor a given analyte. Samples with other types of missing values in anyof the measured analytes were excluded from further analysis.

(II) Data Transformation/Normalization

All analytes were normalized to have a mean of 0 and standard deviationof one across all samples.

(III) Outlier Detection and Removal

No outliers were removed from analysis.

(D) Univariate Analysis

The dataset was split randomly into a training (n=431) and a test set(n=222), stratified by group size. All further analysis except for thevalidation of multivariate decision rules were performed on the trainingset. The univariate analysis comprised the application of parametric andnon-parametric methods to identify individual analytes that wereassociated with the outcome. The false discovery rate was determined toestimate potential biological reproducibility of marker candidates.Analysis of Covariance was used to determine the impact of covariates onthe association between molecular data and outcome. The output of thisanalysis is also used to estimate the usefulness of future subgroupanalysis (e.g. based on covariate-diagnosis interactions).

(E) Multivariate Analysis

Multivariate analysis using Linear Discriminant Analysis (LDA) was aimedat the identification of analyte combinations that were highlyassociated with the outcome variable. Group weights for algorithmtraining were set to be inversely proportional to group size. Stepwisebackward elimination starting with the entire set of 144 analytes wasthen employed to determine the optimal set of analytes. During thisprocedure, every variable was excluded and performance tested by 10 foldcross-validation and quantified using the overall correctness rate(1−error rate). The procedure was stopped when the removal of additionalanalytes did not improve the performance. The model found to be optimalwas then blindly tested on the test set. This showed that the set of 131analytes had an optimal accuracy of 97/98 (% sens/spec MDD vs all),96/99 (% sens/spec SZ vs all) and 97/98 (% sens/spec BD vs all) on thetraining and 74/89 (% sens/spec MDD vs all), 79/86 (% sens/spec SZ vsall) and 63/88 (% sens/spec BD vs all) on the test set.

(F) Subgroup Analysis

The multivariate decision rules may be supplemented with covariates ofinterest or the dataset stratified according to co-variates to estimateseparate decision rules for each covariate level. The same procedure asdescribed in E) may then be repeated.

Results

Plotting the data in the space of the Linear Discriminants (LD) revealedthat LD1 maximized the separation between BD and the remaining twogroups (comparison A) whereas LD2 maximized the separation between SZand the remaining two groups (comparison B). For these comparisons, theindividual variable importance on the training set was determined usingtwo tailed Wilcoxon rank sum tests. This showed that 87 variables weresignificant with p<0.05 for either comparison A or comparison B (seeresults shown in Table 1 wherein the shaded figures show significantresults). A new LDA model was then built in the training set using the87 analytes only and performance tested blindly on the test set. Thisshowed that the set of 87 analytes had an accuracy of 94/96 (% sens/specMDD vs all), 83/95 (% sens/spec SZ vs all) and 97/90 (% sens/spec BD vsall) on the training and 72/89 (% sens/spec MDD vs all), 72/88 (%sens/spec SZ vs all) and 69/83 (% sens/spec BD vs all) on the test set.

TABLE 1 Summary of significant findings

FIG. 1 shows the results obtained when the data was subjected to LinearDiscriminant Analysis (LDA) wherein the x-axis (LD1) separates primarilybipolar disorder from schizophrenia and major depressive disorder andthe y-axis (LD2) separates schizophrenia from bipolar disorder and majordepressive disorder. It will be apparent that combination of both LD1and LD2 provide differential diagnosis between all three groups.

Example 2

This study measured levels of 247 molecules between serum collected from2 separate cohorts. The first cohort contained 35 major depressivedisorder (MDD) patients and 40 well matched controls. The second cohortcontained 40 patients suffering from schizophrenia (paranoid subtype(295.30)) all of which were antipsychotic-naïve or had been offmedication for at least six weeks prior to sample collection and 40 wellmatched controls. Levels of all molecular analytes were determined usinga highly reproducible multiplexed immunoassay platform. The correlationstructure between all analytes was assessed to infer potentialco-regulation structures.

A panel of 15 markers was found to be significantly altered in the MDDgroup when compared with the schizophrenia group. These abnormalitiesremained significant after adjustment for all recorded baselinecharacteristics including age, sex, body mass index and smoking. Amongthe significant markers, a highly prominent correlation structure wasfound.

Methodology Patients

In the present study, samples were investigated from patients sufferingfrom major depressive disorder (MDD) (n=35), schizophrenia (n=40) andwell matched controls (n=40). All individuals were fasted at the time ofblood sample collection and featured no co-morbidities. The ethicalcommittees of the medical faculties of the partner universities approvedthe protocols of this study. Informed consent was given in writing byall participants and clinical investigations were conducted according tothe principles expressed in the Declaration of Helsinki.

Sample Preparation

Blood was collected in S-Monovette 7.5 mL serum tubes (Sarstedt),incubated at room temperature for 2 hours to allow for blood coagulationand then centrifuged at 4000×g for 5 minutes. The supernatant was storedat −80° C. in Low Binding Eppendorf tubes.

Assay Methods

A total of 247 analytes were measured using a set of proprietarymultiplexed immunoassays (Human MAP) at Rules Based Medicine in theirLuminex-based, CLIA-certified laboratory (however measurement couldequally be performed using singleton ELISA). Each antigen assay wascalibrated using 8-point standard curves conducted in duplicate, and rawintensity measurements were interpreted into final proteinconcentrations. Machine performance was verified using quality controlsamples at low, medium, and high levels for each analyte in duplicate.All standard and quality control samples were in a complex plasma-basedmatrix to match the sample background. The autoimmune and infectiousdisease assays were qualitative and the results obtained for unknownsamples were compared with established cut-off values. Because sera wereanalyzed at a previously optimized dilution, any sample exceeding themaximum concentration of the calibration curve was arbitrarily assignedthe concentration of the highest standard, whereas those assayed belowthe minimum concentration of the calibration curve were assigned thevalue 0.0. For analysis, samples were ordered in a manner to avoid anysequential bias due to the presence or absence of disease, patient age,or age of serum sample. Generally, samples alternated between cases andcontrols.

Statistical Analysis

The distribution of the data was examined using standard statistics toassess the necessity for transformations, the presence of outliers orartefactual findings. Parametric (T-test) and non-parametric (WilcoxonRank Sum statistics) univariate methods were applied to identifysignificant differences of molecular levels between the disease andcontrol groups. A p-value of less than 0.05 was considered as beingsignificant. The False Discovery Rate (FDR) was controlled according toBenjamini et al. (J Roy Statist Soc Ser B. 1995; 57:289-300).Multivariate statistics (Principal Component Analysis, PCA and PartialLeast Squares Discriminant Analysis, PLS-DA) were applied to identifypotential groups of markers that discriminated patient from controlgroups and to assess the agreement with univariate methods.

Results

This study investigated levels of 247 molecular analytes in serum from35 patients suffering from major depressive disorder, 40 patientssuffering from schizophrenia and well matched controls (n=40).Demographic details can be found in Table 2:

TABLE 2 Demographic details of patients and healthy volunteers MajorHealthy Depressive Controls Disorder Schizophrenia Number 40 35 40 Sex(m/f) 26/14 13/22 27/13 Age 36 ± 11 40 ± 14 35 ± 10

Applying T-tests, levels of 15 analytes were found to be significantlyaltered between the MDD group and the schizophrenia group (Table 3).These values were in very good agreement with the results obtained fromnon-parametric and multivariate analyses.

TABLE 3 Summary of significant findings Fold Analyte P-value changeProgesterone 0.003515 1.409579 IL-10 0.004413 1.453919 Brain DerivedNeurotrophic Factor 0.013713 0.868194 (BDNF) Serum Amyloid P 0.0148211.228438 sRAGE 0.017552 0.706543 Betacellulin 0.018455 2.267366Chromogranin A 0.022812 1.914507 Creatine Kinase MB 0.024868 1.994627IGF-1 0.02641 0.615415 MIP-3 alpha 0.026793 1.591807 S100b 0.0319041.498588 MMP-3 0.034074 1.390886 IL-1ra 0.035272 1.289866 Tamm-HorsfallProtein (THP) 0.036211 1.348363 IL-18 0.041492 1.209558

In particular, two analytes (MMP-3 and IL-1ra) were also found to besignificantly altered between MDD patients and healthy controls withinthe MDD cohort (Table 4).

TABLE 4 Summary of significant findings Analyte P-value Fold changeMMP-3 0.041043439 0.745898954 IL-1ra 0.006801532 1.334898635

1-19. (canceled)
 20. A method of differential diagnosis forschizophrenia, bipolar disorder, or major depressive disorder or apredisposition thereto in an individual, comprising: a) obtaining abiological sample of an individual; b) quantifying concentrations of apanel of biomarkers in the biological sample; c) generating a biomarkerprofile from the concentrations of the panel of biomarkers of theindividual; d) determining that the biomarker profile of the individualhas a statistically significant similarity with either one of abiomarker profile obtained from healthy control subjects, subjects withschizophrenia, subjects with bipolar disorder, or subjects with majordepressive disorder; and e) classifying the individual as healthy or assuffering from or having a predisposition for either schizophrenia orbipolar disorder or major depressive disorder based at least in part onthe statistically significant similarity of the biomarker profile of theindividual to the biomarker profile of healthy control subjects orsubjects with either schizophrenia, bipolar disorder or major depressivedisorder; wherein the panel of biomarkers comprises one or more analytesselected from Creatine Kinase MB, S100b, or IL-1ra.
 21. The method ofclaim 20, wherein the panel of biomarkers further comprises one or moreanalytes selected from Apolipoprotein H, AXL, BDNF (Brain DerivedNeurotrophic Factor), Betacellulin, Complement 3, CgA (Chromogranin A),EGF R, Endothelin 1, Fas, FGF basic, Haptoglobin, HGF (Hepatocyte growthfactor), Ig A, IL 12p70, IL 10, IL 13, IL 16, IL 1beta, IL 2,Lipoprotein a, MDC, MMP 3, PAI 1, PDGF, Prolactin, Peptide YY, SerumAmyloid P, Thyroxine Binding Globulin, Testosterone, Thyroid StimulatingHormone, TSP 1, or von Willebrand Factor.
 22. The method of claim 21,wherein the panel of biomarkers further comprises one or more analytesselected from Alpha 1 Antitrypsin, ACTH (Adrenocorticotropic Hormone),AgRP (Agouti related Protein), Apolipoprotein A1, BLC (B LymphocyteChemoattractant), Cancer Antigen 125, Carcinoembryonic Antigen,Cortisol, CTGF (Connective Tissue Growth Factor), EN RAGE, Eotaxin,Epiregulin, Erythropoietin, Factor VII, Fas Ligand, Ferritin,Fibrinogen, FSH (Follicle Stimulating Hormone), GM CSF, GST, HB EGF, IFNgamma, IGF-1, Ig M, IL 15, IL 18, IL 1alpha, IL 3, IL 4, IL 5, IL 7,Leptin, LH (Luteinizing Hormone), Lymphotactin, M CSF, MIP 1 alpha, MIP3 alpha, MIP 1 beta, Myoglobin, NrCAM, Prostatic Acid Phosphatase,PAPPA, Progesterone, Prostate Specific Antigen Free, PARC, RANTES,Resistin, SGOT, SHBG, SOD, sRAGE, Tamm-Horsfall Protein (THP), TissueFactor, TECK, TIMP 1, TNF RII, TRAIL R3, or VCAM
 1. 23. The method ofclaim 20, wherein the biological sample comprises cerebrospinal fluid,whole blood, blood serum, plasma, urine, saliva, other bodily fluid,breath, condensed breath, or an extract, purification, or dilution ofany of these.
 24. The method of claim 20, wherein quantifying comprisesSELDI (-TOF) spectrometry, MALDI (-TOF) spectrometry, a 1-D gel-basedanalysis, a 2-D gel-based analysis, mass spectrometry (MS), liquidchromatography (LC), reverse phase liquid chromatography (RP-LC), sizepermeation chromatography, gel filtration chromatography, ion exchangechromatography, affinity chromatography, FPLC, HPLC, UPLC, otherLC-based techniques, other LC-MS-based technique, or combinationsthereof.
 25. The method of claim 20, wherein quantifying comprises animmunological method, a biosensor method, a microanalytical method, amicroengineered method, a microseparation method, animmunochromatography method, or combinations thereof.
 26. The method ofclaim 20, wherein one or more of the biomarkers may be replaced by amolecule, or a measurable fragment of the molecule, found upstream ordownstream of the biomarker in a biological pathway.
 27. A method ofdifferential diagnosis for major depressive disorder, schizophrenia, orhealthy condition in an individual, comprising: a) obtaining abiological sample of an individual; b) quantifying concentrations of apanel of biomarkers in the biological sample; c) generating a biomarkerprofile from the concentrations of the panel of biomarkers of theindividual; d) determining that the biomarker profile of the individualhas a statistically significant similarity with either one of abiomarker profile obtained from healthy control subjects, subjects withschizophrenia, or subjects with major depressive disorder; and e)classifying the individual as healthy or as suffering from or having apredisposition for either schizophrenia or major depressive disorderbased at least in part on the statistically significant similarity ofthe biomarker profile of the individual to the biomarker profile ofhealthy control subjects or subjects with either schizophrenia or majordepressive disorder; wherein the panel of biomarkers comprises one ormore analytes selected from progesterone, IL-10, BDNF (Brain DerivedNeurotrophic Factor), Serum Amyloid P, sRAGE, Betacellulin, CgA(Chromogranin A), Creatine Kinase MB, IGF-1, MIP 3 alpha, S100b, MMP 3,IL-1ra, Tamm-Horsfall Protein (THP), or IL
 18. 28. The method of claim27, wherein the biological sample comprises cerebrospinal fluid, wholeblood, blood serum, plasma, urine, saliva, other bodily fluid, breath,condensed breath, or an extract, purification, or dilution of any ofthese.
 29. The method of claim 27, wherein quantifying comprises SELDI(-TOF) spectrometry, MALDI (-TOF) spectrometry, a 1-D gel-basedanalysis, a 2-D gel-based analysis, mass spectrometry (MS), liquidchromatography (LC), reverse phase liquid chromatography (RP-LC), sizepermeation chromatography, gel filtration chromatography, ion exchangechromatography, affinity chromatography, FPLC, HPLC, UPLC, otherLC-based techniques, other LC-MS-based technique, or combinationsthereof.
 30. The method of claim 27, wherein quantifying comprises animmunological method, a biosensor method, a microanalytical method, amicroengineered method, a microseparation method, animmunochromatography method, or combinations thereof.
 31. The method ofclaim 27, wherein one or more of the biomarkers may be replaced by amolecule, or a measurable fragment of the molecule, found upstream ordownstream of the biomarker in a biological pathway.
 32. A method oftreating an individual suspected of suffering from schizophrenia,bipolar disorder, or major depressive disorder or a predispositionthereto, comprising: a) obtaining a biological sample of an individual;b) quantifying concentrations of a panel of biomarkers in the biologicalsample; c) generating a biomarker profile from the concentrations of thepanel of biomarkers of the individual; d) determining that the biomarkerprofile of the individual has a statistically significant similaritywith either one of a biomarker profile obtained from healthy controlsubjects, subjects with schizophrenia, subjects with bipolar disorder,or subjects with major depressive disorder; e) differentially diagnosingthe individual as healthy or as suffering from or having apredisposition for either schizophrenia or bipolar disorder or majordepressive disorder based at least in part on the statisticallysignificant similarity of the biomarker profile of the individual to thebiomarker profile of healthy control subjects or subjects with eitherschizophrenia, bipolar disorder or major depressive disorder; f)customizing a treatment regimen for the individual based at least inpart on the differential diagnosis; and g) effectuating the treatmentregimen; wherein the panel of biomarkers comprises one or more analytesselected from Creatine Kinase MB, S100b, or IL-1ra.
 33. The method ofclaim 32, wherein the panel of biomarkers further comprises one or moreanalytes selected from Apolipoprotein H, AXL, BDNF (Brain DerivedNeurotrophic Factor), Betacellulin, Complement 3, CgA (Chromogranin A),EGF R, Endothelin 1, Fas, FGF basic, Haptoglobin, HGF (Hepatocyte growthfactor), Ig A, IL 12p70, IL 10, IL 13, IL 16, IL 1beta, IL 2,Lipoprotein a, MDC, MMP 3, PAI 1, PDGF, Prolactin, Peptide YY, SerumAmyloid P, Thyroxine Binding Globulin, Testosterone, Thyroid StimulatingHormone, TSP 1, or von Willebrand Factor.
 34. The method of claim 33,wherein the panel of biomarkers further comprises one or more analytesselected from Alpha 1 Antitrypsin, ACTH (Adrenocorticotropic Hormone),AgRP (Agouti related Protein), Apolipoprotein A1, BLC (B LymphocyteChemoattractant), Cancer Antigen 125, Carcinoembryonic Antigen,Cortisol, CTGF (Connective Tissue Growth Factor), EN RAGE, Eotaxin,Epiregulin, Erythropoietin, Factor VII, Fas Ligand, Ferritin,Fibrinogen, FSH (Follicle Stimulating Hormone), GM CSF, GST, HB EGF, IFNgamma, IGF-1, Ig M, IL 15, IL 18, IL 1alpha, IL 3, IL 4, IL 5, IL 7,Leptin, LH (Luteinizing Hormone), Lymphotactin, M CSF, MIP 1 alpha, MIP3 alpha, MIP 1 beta, Myoglobin, NrCAM, Prostatic Acid Phosphatase,PAPPA, Progesterone, Prostate Specific Antigen Free, PARC, RANTES,Resistin, SGOT, SHBG, SOD, sRAGE, Tamm-Horsfall Protein (THP), TissueFactor, TECK, TIMP 1, TNF RII, TRAIL R3, or VCAM
 1. 35. The method ofclaim 34, wherein the treatment regimen is further customized based atleast in part on whether there is a statistically significant similarityor difference between the individual's biomarker profiles generated ontwo or more occasions.
 36. The method of claim 35, wherein biomarkerprofiles from the individual are generated prior to commencement of thetreatment regimen, and/or at an earlier stage of the treatment regimen.37. The method of claim 35, wherein biomarker profiles from theindividual are generated at intervals throughout the individual'slifetime.
 38. The method of claim 34, wherein customizing the treatmentregimen further comprises one or more selections of therapy selectedfrom: type and/or dosing of medications, type and/or duration ofpsychotherapy, type and/or duration of counseling, type and/or durationof hospitalization, type and/or dosing of electroconvulsive therapy. 39.A kit for differentially diagnosing a healthy condition, schizophrenia,bipolar disorder or major depressive disorder, or predisposition for ahealthy condition, schizophrenia, bipolar disorder or major depressivedisorder, comprising a biosensor capable of quantifying MMP 3, Alpha 1Antitrypsin, ACTH (Adrenocorticotropic Hormone), AgRP (Agouti relatedProtein), Apolipoprotein A1, Apolipoprotein H, AXL, Betacellulin, BLC (BLymphocyte Chemoattractant), BDNF (Brain Derived Neurotrophic Factor),Complement 3, Cancer Antigen 125, Carcinoembryonic Antigen, CgA(Chromogranin A), Creatine Kinase MB, Cortisol, CTGF (Connective TissueGrowth Factor), EGF R, Endothelin 1, EN RAGE, Eotaxin, Epiregulin,Erythropoietin, Factor VII, Fas, Fas Ligand, Ferritin, FGF basic,Fibrinogen, FSH (Follicle Stimulating Hormone), GM CSF, GST,Haptoglobin, HB EGF, HGF (Hepatocyte growth factor), IFN gamma, IGF-1,Ig A, Ig M, IL 10, IL 12p70, IL 13, IL 15, IL 16, IL 18, IL 1 alpha, IL1beta, IL 1ra, IL 2, IL 3, IL 4, IL 5, IL 7, Leptin, LH (LuteinizingHormone), Lipoprotein a, Lymphotactin, M CSF, MDC, MIP 1 alpha, MIP 3alpha, MIP 1beta, Myoglobin, NrCAM, PAI 1, Prostatic Acid Phosphatase,PAPPA, PDGF, Progesterone, Prolactin, Prostate Specific Antigen Free,PARC, Peptide YY, RANTES, Resistin, S100b, Serum Amyloid P, SGOT, SHBG,SOD, sRAGE, Tamm-Horsfall Protein (THP), Thyroxine Binding Globulin,Testosterone, Tissue Factor, TECK, TIMP 1, TNF RII, TRAIL R3, ThyroidStimulating Hormone, TSP 1, VCAM 1, and von Willebrand Factor.